Apoptosis Detection Kits: Tools for Apoptosis-Inducing Potentcy Evaluation | Biowire Spring 2012

By: Dalit Weinstein-Fischer, Ph.D., Supervisor of Research & Development; Rina Altman, M.Sc., Scientist of Research & Development; Dorit Zharhary, Ph.D., Director of Research & Development, Biowire Spring 2012, 22–24

Biowire Spring 2012 — Live Cell Imaging of Signaling Pathways

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Programmed cell death is a fundamental process important in development, as well as a principle mechanism of tumor suppression. Apoptosis is triggered in non-malignant cells as a protective mechanism to remove damaged and unhealthy cells that may harm the body. Many chemotherapy treatments are based on the induction of programmed cell death1,2. Cells undergoing apoptosis are morphologically distinct from necrotic or autophagic cell death. Cells dying via apoptosis are characterized by cell shrinking, nuclear condensation and fragmentation, membrane blebbing, and finally separation of the cellular components into apoptotic bodies, which are then engulfed by phagocytes.

Apoptosis is thought to occur through two main pathways: the intrinsic pathway where the death signal arises from within the cell, and the extrinsic pathway, which involves the activation of cell surface receptors by an extracellular death factor. The intrinsic pathway can be triggered by various intracellular stimuli including DNA damage, growth factor starvation, and oxidative stress. These signals lead to mitochondria outer membrane permeability, resulting in mitochondrial protein leakage, which in turn induces apoptosis through activation of members of the cysteine-aspartic acid protease (caspase) family. Caspases are found in the cell as inactive proenzymes, cleavage of which produces active proteases3. For the intrinsic pathway, the initial enzyme activated is caspase-9, followed by the activation of caspase-3, 6, and/or 7. The extrinsic apoptotic pathway is stimulated by binding of a death ligand to a specific receptor, initiating signaling leading to caspase-8 activation. Caspase-8 subsequently either directly activates caspase-3 or induces mitochondria outer membrane permeability to trigger the cascade of caspases-3, 6, and/or 7, leading to cell death4.

In this study, we analyzed the ability of three small molecule antibiotics derived from specific microorganisms, to induce apoptosis in Jurkat cells. These included Ikarugamycin (Cat. No. SML0188), Siomycin A (Cat. No. S6076), and Zapotin (Cat. No. Z4652), all previously reported in the literature as apoptosis inducers5,6,8. For assaying apoptosis, we used two different apoptosis detection kits, Annexin V-FITC Apoptosis Detection Kit (Cat. No. APOAF) and Caspase 3 Assay Kit, Fluorimetric (Cat. No. CASP3F), which detect apoptosis at different stages.

The early stages of apoptosis involve cellular changes that include loss of phospholipid asymmetry. At the onset of apoptosis, phosphatidylserine translocates from the internal to the external side of the plasma membrane. The phosphatidylserine thus becomes available to bind to Annexin V in the presence of calcium. The Annexin V-FITC Apoptosis Detection Kit detects apoptotic cells by flow cytometry utilizing Annexin V-FITC as a fluorescent probe to detect early apoptotic phosphatidylserine binding. Propidium iodide binding to DNA is also monitored to indicate compromise of the cell membrane and progression in apoptosis.

The Caspase 3 Fluorimetric Assay Kit measures the level of caspase-3 activity in cell lysates. The kit is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD -AMC) by caspase-3, resulting in the release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety.

Methods and Results Analysis

Jurkat cells were grown to a density of 1 x 106 cells/mL and were either left untreated or treated with increasing concentrations of the following antibiotics: Zapotin (Cat. No. Z4652), Siomycin A (Cat. No. S6076), and Ikarugamycin (Cat. No. SML0188). Cells were then analyzed for apoptosis induction using two methods:

  1. Staining with Annexin V: After 24 hours of incubation with the indicated molecules, cells were washed and stained with Annexin V-FITC Conjugate and Propidium Iodide (PI) using Apoptosis Detection Kit (Cat. No. APOAF) following the kit technical bulletin, and analyzed by flow cytometry. The level of apoptosis induction is assessed by depicting cell staining by PI (y-axis) vs Annexin V (x-axis) (Figure 1). The lower left quadrant (PI and Annexin V negative cells) shows the percentage of live cells. On the lower right quadrant are cells stained with only Annexin V, which are cells undergoing early apoptosis. Cells stained by both Annexin V-FITC and PI (top right quadrant) are cells undergoing late apoptosis/necrosis. Cells stained with only PI (upper left quadrant) are necrotic/non-viable cells.
  2. Caspase-3 Activity: Cells were incubated for 4, 8, or 24 hours with the above mentioned molecules, washed, and analyzed for caspase-3 activity using the Caspase 3 Assay Kit, Fluorimetric (Cat. No. CASP3F) following the kit technical bulletin (Figure 2). Results are presented as fluorimetric units per mg protein.
Apoptosis Induction with Ikarugamycin, Zapotin or Siomycin A

Figure 1. Apoptosis Induction with Ikarugamycin (Cat. No. SML0188), Zapotin (Cat. No. Z4652), or Siomycin A (Cat. No. S6076). Jurkat cells were left untreated (A), or treated for 24 hours with one of the following: 1 µM Ikarugamycin (B), 5 µM Ikarugamycin (C), 10 µM Zapotin (D), 20 µM Zapotin (E), 5 µM Siomycin A (F), or 10µM Siomycoin A (G). Cells were stained with Annexin V-FITC Conjugate and Propidium Iodide (PI) using Apoptosis Detection kit (Cat. No. APOAF) and analyzed by flow cytometry. Cells, which were stained by Annexin V-FITC, are cells in early apoptosis (bottom right quadrant), while cells stained with both Annexin V-FITC and PI (top right quadrant) are cells undergoing late apoptosis (FL1, Annexin V-FITC; FL2, PI).

Kinetics of Apoptosis Induction by Siomycin A, Ikarugamycin or Zapotin

Figure 2. Kinetics of Apoptosis Induction by Siomycin A (Cat. No. S6076), Ikarugamycin (Cat. No. SML0188), or Zapotin (Cat. No. Z4652). Jurkat cells were untreated or treated for 4 hours (orange), 8 hours (blue), and 24 hours (red) with increasing concentrations of Ikarugamycin, Zapotin, or Siomycin A. Cells were monitored for their caspase-3 activity using Caspase 3 Assay Kit, Fluorimetric (Cat. No. CASP3F), and analyzed by a fluorimeter. Results are normalized to the untreated cells and presented as fluorimetric units per mg protein.

Results and Conclusions

After testing these two methods of apoptosis detection, it is evident from the data shown that all three antibiotics tested are potent apoptosis inducers (Figure 1 and Figure 2). Cells treated with 1 μM of Ikarugamycin for 24 hours showed induction of apoptosis with 38% of the cells being in late apoptosis (Figure 1B). Elevating the concentration of Ikarugamycin increased apoptosis induction resulting in nearly complete apoptosis in all cells (98%, Figure 1C). Treatment of Jurkat cells with either 10 μM or 20 μM of Zapotin induced apoptosis in 46% and 54% of the cells, respectively (Figures 1D and 1E), while Siomycin A treatment with 5 μM and 10 μM induced apoptosis in 55% and 51% of the cells, respectively (Figures 1F and 1G).

Since all three antibiotics induced apoptosis at different potency following 24 hours of treatment, we wanted to test whether this difference may not be detected if tested at different induction times. Therefore, Jurkat cells were treated for 4, 8, or 24 hours. As shown in Figure 2, Jurkat cells treated with each of the antibiotics for the indicated times and concentrations underwent apoptosis. Interestingly, apoptosis kinetics diverted between the various antibiotics as Ikarugamycin reached its maximum activity at 24 hours post-treatment with 5 µM, while Siomycin A maximal induction was detected 8 hours post-treatment with 5 µM. However, treatment with 50 µM Zapotin for 8 hours seemed to induce caspase-3 activity more efficiently than treatment with the other antibiotics tested (Figure 2).


In this study, we wanted to compare the ability of three antibiotics to induce apoptosis. We found that, under the tested conditions, all three antibiotics induced apoptosis in Jurkat cells. However, they differed in the kinetics of their activity and in their intensity, with Zapotin and Ikarugamycin being the more potent inducers.

The apoptotic activity of the three antibiotics has been previously demonstrated to induce cleavage of caspase-9, 8, and 3 in treated cells. Monitoring of intracellular calcium levels by flow cytometry analysis indicated an increase in cytosolic calcium levels that correlated with the cleavage of caspases5. Ikarugamycin was analyzed for its DNA damage induction by monitoring γH2AX phosphorylation, indicating the activation of the intrinsic apoptotic pathway.

Siomycin A was found to activate apoptosis in NB7 cells which are caspase-8-deficient mutants that could not undergo extrinsic apoptosis. Nevertheless these cells were almost as sensitive to the Siomycin A treatment as reconstituted cells, suggesting that Siomycin A-induced apoptosis mainly involves the intrinsic apoptotic pathway6,7. Siomycin A was also found to inhibit the transcriptional activity of FoxM1, which is an oncogenic transcription factor that is activated in most human tumors. Researchers observed direct correlation between FoxM1 inhibition and caspase-3 cleavage that occurred 18 hours post-treatment with 10 μM Siomycin A6,7.

Treatment with Zapotin induced apoptosis in a wide range of cancer cell lines such as colon cancer, hepatocarcinoma, nasopharyngeal carcinoma, breast adenocarcinoma, and epithelial carcinoma cells. 10 μM treatment in Hep-G2 cells induced chromatin condensation, nuclear disassembly, and DNA fragmentation. In addition, in these cells both caspase-8 and caspase-9 activity was significantly increased, which in turn activated caspase-3 activity. Also, pro-apoptotic BAX was up-regulated8,9. These results are in accordance with the documented ability of these molecules to induce apoptosis.




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  4. Long JS, Ryan KM. New frontiers in promoting tumor cell death: targeting apoptosis, necrosis and autophagy. Oncogene, advanced online publication. 6 February 2012. 28 February 2012 (http://www.nature.com/onc/journal/vaop/ncurrent/full/onc20127a.html).
  5. Popescu R, et al. Ikarugamycin induces DNA damage, intracellular calcium increase, p38 MAP kinase activation and apoptosis in HL-60 human promyelocytic leukemia cells. Mutat Res. 2011; 709-710:60–66.
  6. Bhat UG, et al. FoxM1 is a general target for proteasome inhibitors. PLoS One. 2009;4:e6593.
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